The majority of Parkinson's disease (PD) occurs sporadically without any autosomal inheritance patterns. During the first funding period, the UVA Udall Center group accumulated substantial circumstantial evidence that mitochondria! genomes contribute significantly to PD pathogenesis. This evidence included spontaneous production of true Lewy body inclusions in PD cybrids derived from PD platelet mtDNA. Although a previous project has identified mtDNA mutations in complex I genes that segregate in and are predictive of PD brain, direct proof of causality of PD brain mtDNA mutations in driving PD pathogenesis is lacking. In addition, questions about the relevance of platelet mtDNA to brain pathology continue to plague interpretation of cybrid studies. These problems are directly addressed in this project. This Project utilizes novel technologies developed recently in the Bennett lab that allow rapid removal of, and insertion and expression of the entire human mitochondrial genome into mitochondria of living cells. The development of an engineered mitochondrial DNA-binding protein incorporating a protein transduction domain ("protofection") allows the creation of cell lines expressing gel-purified mtDNA from human postmortem brain. Characterizing the phenotypes of these cell lines will allow a direct test of causality of brain mtDNA in driving the pathogenesis of Parkinson's disease. The Bennett group has also successfully engineered mtDNA to incorporate point mutations and expression of exogenous genes specifically in the mitochondrial compartment. This achievement, in combination with protofection technology, will allow the creation of mutated mtDNA species incorporating complex I gene mutations associated with PD brain. The pathogenecity of these mutant mtDNA species can now be tested directly by expressing these mtDNA species in cell lines. There are four Aims in this Project. Aim 1 will optimize protofection technology in order to create neural cell lines from expression of mtDNA purified from PD and CTL brains classified according to Braak staging. Bioenergetic activity, oxidative stress markers and proteasomal function/protein levels will be compared between the brain samples of origin and the cell lines resulting from expression of mitochondrial genomes derived from those brain samples. Aim 2 will determine phenotypes of neural cells created by protofection to express homoplasmically mitochondrial genomes altered to contain mutations discovered in Project 1 to be specific to PD brain. Aim 3 will study heteroplasmy that is created in neural cell lines and will test the hypothesis that PD pathogenic mtDNA mutations have a replicative advantage. Aim 4 will develop cell models for mitochondrial gene replacement therapy of pathogenic PD mtDNA mutations. These Aims will provide highly interpretable outcomes to support or refute the involvement of brain mtDNA in PD pathogenesis, will define pathogenecity of specific mtDNA complex I gene mutations and will set the stage for development of mitochondrial gene replacement therapy.